@PhdThesis{diss_mods_00015665, author = {Sch{\"o}nbeck, Benjamin John}, title = {Proteome analyses for the characterization of the ADAM17ex/ex hypomorphic mouse model}, year = {2014}, publisher = {Christian-Albrechts-Universit{\"a}t zu Kiel}, address = {Kiel}, keywords = {2D-DIGE; ADAM17; MEF}, abstract = {The ectodomain shedding of numerous transmembrane proteins by the A Disintegrin And Metalloprotease 17 (ADAM17) governs a variety of physiological and developmental processes. Focusing on murine embryonic fibroblasts (MEFs) of ADAM17wt/wt and ADAM17ex/ex (hypomorphic) mice, we investigated ADAM17-mediated changes in conditioned cell culture supernatants, in membrane preparations and in whole cell lysates using a 2D-DiGE proteomic approach. The detection of high amounts of cleaved TNF$\alpha$ in supernatants of TNF$\alpha$-overexpressing wild-type but not ADAM17ex/ex MEF cells demonstrated the general suitability of the employed 2D-DiGE approach for the identification of shed protein fragments. Interestingly, the analyses of whole-cell lysates revealed that ADAM17 might also influence intracellular protein levels since a variety of proteins (e.g. annexins) showed altered abundances at reduced levels of ADAM17. From conditioned MEF supernatants, seventy differentially abundant secreted and cell-surface proteins were identified. These included collagens and other extracellular matrix components, several proteases and several protein fragments. Thus, wild-type and ADAM17ex/ex MEF cells clearly differ in their ability to secrete or shed proteins. However, only fourteen different protein fragments showed altered abundances in ADAM17ex/ex MEF cell supernatants, including only two known ADAM17 substrates (TNF$\alpha$ and NCAM-1). Notably, most full length proteins that were detected as fragments lack a transmembrane region and are thus no bona fide ADAM17 substrates. The high amounts of the perlecan LG3 peptide and of the type-I collagen C-terminal propeptide in supernatants of wild-type MEF cells point to a reduced collagen maturation and degradation in ADAM17ex/ex MEF cells. Since we also observed reduced levels of the C-procollagenase ``bone morphogenic protein 1'' and reduced matrix metalloproteinase activity in ADAM17ex/ex MEF cell culture supernatants, the observed processing of collagen and perlecan might reflect ``proteolytic side effects'' by other proteases in a complex ``protease web''.}, url = {https://macau.uni-kiel.de/receive/diss_mods_00015665}, file = {:https://macau.uni-kiel.de/servlets/MCRFileNodeServlet/dissertation_derivate_00005694/Schoenbeck_Thesis.pdf:PDF}, language = {en} }